The mission of Novab is to utilize the unique properties of lamprey generated Variable Lymphocyte Receptors (VLRs) to develop effective, complementary and/or superior therapeutics to those currently derived from mammalian monoclonal antibodies (mMAbs). In the current application, we seek to initiate and demonstrate proof of concept for one commercial project within this mission, which is the application of VLRs to the design of chimeric antigen receptors (CARs) that currently are used as the tumor targeting component of genetically- engineered T cell immunotherapies. Our hypothesis is that due to independent evolution from the immunoglobulins (Igs) of jawed vertebrates, lamprey VLRs raised against therapeutic cell targets will recognize epitopes that are unique from those obtained using standard mammalian immunization and MAb (i.e. Ig) production. Therefore, VLRs represent a novel platform for the identification of cancer cell targets and the generation of improved diagnostics and biotherapeutics (e.g. monoclonal VLRs or VLR-CARs). Recently, CARs expressing T cells (CARTs) have demonstrated a high degree of efficacy in a limited set of otherwise refractory cancers. Currently, CAR diversity is restricted by the identification and availability of effective MAb binding domains, which in turn is restricted by not only mammalian genetic diversity but also by Ig structural geometry and pre-existing immune tolerance barriers to most antigens of human origin. We predict that VLR technology will allow for more specific cancer cell targeting and appreciably expand the range of cancer types that can be effectively treated using CARTs. Specifically, we will test this prediction using two independent hematopoietic tumor models. Using high-throughput screening and molecular cloning technology, tumor- specific VLRs were generated against a mouse BCL tumor cell line and primary human multiple myeloma cells. These VLRs will be utilized as the antigen binding components of novel CARs. We will demonstrate if VLR- CARs can be used as viable anti-tumor treatments using a series of in vitro tests (Milestone 1) and immune- competent and immune-compromised mice (Milestone 2). Completion of the Milestones presented in this proposal will provide the fundamental proof of concept evidence that VLR-CARTs are a scientifically-viable platform for the development of effective anti-cancer biotherapeutics.